Truseq dna sample preparation

Exome enrichment was performed with Illumina's TruSeq DNA Sample Preparation and sequenced on the Illumina HiSeq 1500 platform. FASTQ files were aligned to the human reference genome GRCh37 (feb.2009) using the Novoalign v. 3 algorithm (http://www.novocraft.com) at default parameters. Removal of duplicate reads, recalibration and local realignment around indels was performed using Best Practices pipeline v. 2.7 [30 (link)]. The result was mean coverage rates in the exome region between 89 x and 148 x (Supplementary Table 1).

Soil DNA was extracted according to Mandakovic et al. (2018a (link)) with some modifications. Total DNA from soils was extracted from 10 g of each sample using the NucleoSpin® Food kit (Macherey-Nagel), following the manufacturer's instructions, and the CTAB extraction buffer published by Zhou et al. (1996 (link)). Microbial 16S rRNA genes were amplified according to Mandakovic et al. (2018b (link)) without modifications. DNA libraries were constructed following the TruSeq DNA sample preparation (Illumina, USA) protocol. Sequencing was performed by MrDNA Next Generation Sequencing Service Provider (Shallowater, Texas, USA) on an Illumina MiSeq platform in an overlapping 2 × 300 bp configuration, to obtain a minimum throughput of 40,000 sequences (reads) per sample.

One microgram of genomic DNA from each sample was randomly fragmented by focused acoustic shearing (Covaris inc.) according to Illumina’s protocol. The fragment length was measured by Bioanalyzer (Agilent Technologies 2100), confirming a fragment length of 150–300 bp. Exome enrichment was performed with Illumina’s TruSeq DNA Sample Preparation. Paired end sequencing of 2 x 100 bases was performed on the Illumina HiSeq 1500 platform. FASTQ files were aligned to the human reference genome GRCh37 (feb.2009) using the Novoalign v. 3 algorithm (www.novocraft.com) at default parameters. Removal of duplicate reads, recalibration and local realignment around indels were performed using Best Practices pipeline v. 2.7 [15 (link)]. The result was a mean coverage rate in the exome region of 65–155 x in the tumor samples and 11–148 x in the matched normal samples (S1 Table).

Genomic DNA was extracted from whole flies using the DNeasy Blood and Tissue DNA Extraction kit (Qiagen Inc., Valencia, CA) and pooled from five female and five male Phormia regina flies housed in a laboratory colony (approximately 4–6 months old). The founders originated from Indianapolis, IN (39.7684° N, 86.1581° W) and were collected during the summer of 2012. The extracted DNA from each individual was quantified using a Qubit fluorometer (ThermoFisher Scientific, Grand Island, NY) and mixed in equal proportions to yield the two pooled extracts, one for each sex. DNA libraries were prepared using TruSeq DNA sample preparation (Illumina, San Diego, CA) and sequenced (2 × 100 bp) using one half lane of an Illumina HiSeq2000 platform at the Purdue University Genomics Core Facility (West Lafayette, IN). Additional 454 reads were obtained as described in [44 (link)].

Microbiome DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions and including mechanic lysis of the samples using disruption spheres (FastPrep-24 MP). Extracted DNA was visualized in Tape Station 2200 (Agilent Technologies, Santa Clara, CA, USA) using Genomic DNA Screen Tape, according to the manufacturer’s indications and quantified by fluorescent probes (Qubit Thermo Fisher Scientific, Waltham, MA, USA).
Internal transcribed spacer (ITS) was amplified using the primer set ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’), with a barcode in the forward primer. For the amplification, the kit HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany) was used with the following conditions: 94 °C 3 min, 28 cycles of 94 °C 3 s, 53 °C 4 s, and 72 °C 1 min, followed by an elongation phase of 72 °C 5 min. PCR products were examined in agarose gels (2%). Samples were purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA). DNA libraries were constructed following the protocol TruSeq DNA sample preparation (Illumina, San Diego, CA, USA). Sequencing was performed by MrDNA Next Generation Sequencing Service Provider (Shallowater, TX, USA) on Illumina MiSeq platform in an overlapping 2 × 300 bp configuration to obtain a minimum throughput of 20,000 sequences (reads) per sample.